Preparation of 2-chloro-7-hydroxy-11-(4-methyl-1-piperazinyl)- di benz(b,f)-oxazepine

ABSTRACT

A method for converting 2-chloro-11-(4-methyl-1piperazinyl)dibenz(b,f)(1,4)oxazepine to 2-chloro-7-hydroxy-11(4-methyl-1-piperazinyl)dibenz(b,f)(1,4)oxazepine through fermentative biosynthesis employing the organism Cunninghamella elegans. The compound is physiologically active as a tranquilizer and antidepressant.

United States Patent McGahren et al.

[ Nov. 13, 1973 PREPARATION OF 2-CHLORO-7-HYDROXY-l l-(4-METHYL-l-P1PERAZ1NYL)- DI BENZ(B,F)-OXAZEPINE Inventors: William James McGahren,

Demarest; Martin Paul Kunstmann, Pearl River, both of NY.

Assignee: American Cyanamid Company,

Stamford, Conn.

Filed: Sept. 29, 1972 Appl. No.: 293,711

US. Cl 195/51 R Int. Cl C12d 13/00 Field of Search 195/51 R ReferencesCited UNITED STATES PATENTS 5/1967 Nielson et a1. 195/51 R 3,453,1797/1969 Greenspan et a1. 195/51 R Primary Examiner-Alvin E. TanenholtzAttorney-Ernest Y. Miller [5 7] ABSTRACT 4 Claims, No DrawingsPREPARATION OF 2-CHLORO-7-HYDROXY-I I-( 4-METH Y I -L. PIPERAZINYL)- DIBENZ(B,F)-OXAZEPINE PRIOR ART A method for the preparation of2-chloro-ll-(4- methyl-l-piperazinyl)dibenz[b,f][1,4]oxazepine isdescribed and claimed in U. S. Pat. No. 3,444,169. This compound is alsodescribed and claimed in a method for the control of fertility inwarm-blooded female animals in U. S. Pat. No. 3,412,193.

The product resulting from the present fermentation,2-chloro-7-hydroxy-l l-(4-methyl-lpiperazinyl)dibenz[b,f][l,4]-oxazepine, is specifically described and claimed in U. S. Pat. No.3,660,406.

DESCRIPTION OF THE INVENTION The compound 2-chloro-ll-(4-methyl-lpiperazinyl)dibenz[b,f][1,4]oxazepine (I) is converted to2-chloro-7-hydroxy-l l(4-methyl-l-piperazinyl)-dibenz[b,f][l,4]oxazepine (II), a highly effective psychoactive agent inan aerobic fermentation procedure employing the microorganismCunninghamella elegans (ATCC 9245). The reaction which takes place canbe This microbiological method represents an improvement over thechemical conversion process for preparing compound (II) in that thechemical conversion is a multistep process with a comparatively lowoverall yield.

It was found that all of the cultures screened, which amounted to atotal of 60, including eight strains of Cunninghamella elegans, onlyATCC 9245 was capable of conducting the subject transformation.

FERMENTATION PROCESS Cultivation of the organism Cunninghamella elegansATCC 9245 obtained from the American Type Culture Collection may becarried out in a wide variety ofliquid culture media. Useful media mayinclude an assimilable source of carbon such as starch, sugar, molasses,glycerol, etc.; an assimilable source of nitrogen such as protein,protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.;and inorganic anions and cations, such as potassium, sodium, calcium,sulfate, phosphate chloride, etc. Trace elements such as boron,molybdenum, copper, etc.; are usually supplied as impurities of otherconstituents of the media. Aeration in tanks or bottles is provided byforcing sterile air through or'onto the surface of the fermentingmedium. Further agitation in tanks is provided by a mechanical impeller.An antifoaming agent such as lard oil may be added as needed.

INOCULUM PREPARATION Flask inoculum of Cunninghamella elegans (ATCC9245) is prepared by inoculating ml. of sterile liquid medium in 250 ml.flasks with scrapings or washings of spores from an agar slant of theculture. The following is an example of a suitable medium:

Corn steep liquid 2% Glucose -4% KH PO 0.6%

CaCO 0.5%

Water to 100% pH adjusted to 6.2

The flasks are incubated at 20 C. on a rotary shaker for 72 hours.

FERMENTATION PROCEDURE Aliquots of about 5 ml. of the above describedinoculum are then used to inoculate flasks containing sterilefermentation medium. A suitable medium is exemplified below:

Corn steep liquor 2% Glucose 2% Na HPO, 0.6%

CaCO 0.5%

Water to -l00% pH adjusted to 6.2

The flasks are incubated for 24 hours at which time 2-chloro-ll-(4-methyll -piperazinyl)dibenz[b,f][ l 4]oxazepine in methanol isadded to the fermentation flask at an approximate concentration of 200mg. per liter of medium. The fermentation is continued for an additional-l20 hours.

ISOLATION PROCEDURE The pH of the harvest mash is adjusted to 7.0 andextracted with an equal volume of n-butanol. The butanol extract isconcentrated to a gummy residue. This residue is passed through acidwashed diatomaceous earth using the system hexane:- ethylacetate:methanol:water (:30:15z6). Appropriate fractions from thepartition column are combined and concentrated to an oil suspensionwhich upon trituration with ether leaves a gray residue. Furthertrituration of this residue with ether produces an off-white solid whichis dissolved in methanol and applied to a Brinkmann 2 mm. thick layerplate and developed using the system chloroformzmethanol 8:2. The morepolar band is eluted batchwise to give a residue which is againtriturated with ether yielding 2- chloro-7-hydroxy-ll-(4-methyl-l-piperazinyl)- dibenz[b,f][1,4]oxazepine as a white solid.

SPECIFIC DISCLOSURE The present invention is further described by thefollowing specific examples:

EXAMPLE I Shaker Flask Transformation of I to II Using (ATCC 9245) Two250 ml. Erlenmyer flasks each containing 50 ml. of the following sterilemedium:

Corn steep liquor 2% Glucose 4% Water to l% are adjusted to pH 6.2 andinoculated with spores from an agar slant of Cunninghamella elegans(ATCC 9245). These flasks are incubated at 20 C. for 72 hours on arotary shaker. Between 3 and ml. of this inoculum is then added to eachof 22 Erlenmyer flasks each containing 50 ml. of the following sterilemedium:

Corn steep liquor 2% Glucose -2% Na HPO. 0.6%

CaCO O.5%

Water to l00% The pH is adjusted to 6.2 and incubation is continued for24 hours. At this time mg. of 2-chloro-l l-(4-methyl-l-piperazinyl)dibenz[b,f][ l ,41oxazepine in 0.] ml. of methanolis added to each flask. Fermentation is continued under the sameconditions. The flasks are harvested 64 hours after substrate addition.The mashes are combined and the pH is adjusted to 7.0. The mash isextracted with an equal volume of nbutanol. The butanol is concentratedto a gummy residue of 560 mg. which is passed through 75 gm. of acidwashed diatomaceous earth using the system hexanezethylacetate:methanol:-water (70:30:1526). The volume of each of the first 21fractions is 3035 ml. and the remaining fractions are in the range 60-65ml. Fractions 25 through 37 are combined and concentrated to 250 mg. ofoily suspension, which upon trituration with ether gives 150 mg. of grayresidue. The 140 mg. is again triturated with ether and the residueprovides 60 mg. of off-white solid. This solid is dissolved in a smallamount of methanol and applied to a Brinkmann 2 mm. thick layer plateand developed using chloroform:methanol (8:2). The more polar band ofthe two main bands is eluted batchwise yielding 30 mg. of a residuewhich is again triturated with ether leaving 12 mg. of white solid. Theinfrared spectrum of this product is identical with that of authentic2-chl0ro- 7-hydroxy-l l-(4-methyll -piperazinyl dibenz[b,f][ l,4]oxazepine.

EXAMPLE 2 Tank Transformation of l to ll Using (ATCC 9245) A slant ofCunninghamella elegans (ATCC 9245) is used to inoculate 50 ml. of theinoculum medium described in Example I. The medium is incubated at C.for 72 hours on a rotary shaker. Approximately 5 ml. aliquots of thisinoculum are added to each of four 250 ml. Erlenmyer flasks containing50 ml. each of the same inoculum medium. After 48 hours of growth these4 flasks are used to inoculate 2 liters of the same inoculum medium inan air-agitated bottle. This medium is incubated for 48 hours, Thecontents of the bottle are added to a 30 liter tank containing 20 litersof the fermentation medium described in Example l. After 24 hours oftank growth 5.0 gm. of 2-chloro-l l-(4-methyl-l-piperazinyl)dibenz[b,f][1,4]oxazepine in 35 ml. of methanolis added to the tank. The tank is harvested 70 hours after substrateaddition. The mash is adjusted to pH 7.0 and extracted with 1/2 of itsvolume of n-butan0l. Concentration of the n-butanol extract yields 30gm. of crude black oil. Trituration of this oil with ether gives 21 gm.of a gummy solid which is taken up in lOO ml. of methanol and thenconcentrated to about ml. The addition of 250 ml. of ether to thismethanolic solution gives a gummy precipitate which is washed with etherand dried to yield 16 gm. This material is passed through 600 gm. ofacid washed diatomaceous earth using the system hexane:ethylacetate:methanol:water (:40:20z6). Fractions 66 through 126, having anaverage fraction volume of -85 ml., are combined to yield 2.0 gm. ofbrown oil which is dried over phosphorus pentoxide for 16 hours to yield1.7 gm. Trituration of this material with ether yields 1.] gm. of lightbrown solid which by ultraviolet analysis is 62% 2-chloro-7-hydroxy-ll-(4-methyl -l-piperazinyl)- dibenz[b,f][ l,4]oxazepine.

EXAMPLE 3 Tank Transformation ofl TO ll Using (ATCC 9245) The procedureof Example ll is repeated. The mash is harvested 29 hours aftersubstrate addition and the pH is adjusted to 7.0. Extraction of the mashwith I12 its volume of n-butanol and concentration of the nbutanol givesan oil which is partitioned between methanol and heptane. Concentrationof the methanolic phase gives 15 gm. ofa gum which is chromatographedover 600 gm. of acid washed diatomaceous earth using the systemhexanezethyl acetatezmethanolzwater (6014011516). Combination offractions 67 to 130, average fraction volume 80-85 ml. gives 3.5 gm. ofan oil which is refrigerated overnight and then triturated with ether toyield 2.2 gm. of light brown solid which is about 76% pure with regardto the desired derivative. A 60 mg. portion of this material isdissolved in 0.3 ml. of methanol and applied to a Brinkmann 2.0 mm.silica gel thick layer plate and developed using chloroform- :methanol(8:2). The more polar main band is scraped off and eluted in a columnwith 20 ml. of 50:50 chloroform-methanol solution to obtain 30 mg. offaintly yellow solid. Upon trituration of this solid with ether ll mg.of off-white material is obtained which by infrared analysis isidentical with 2-chloro-7-hydroxy-l l-(4-methyl-l-piperazinyl)dibenz[b,f}[1,4]oxazepine.

We claim:

1. A method of preparing 2-chloro-7-hydroxy-l I-{4-methyl-l-piperazinyl)dibenz[b,f][1,4]oxazepine which comprisessubjecting 2-chlorol l-(4-methyll piperazinyl)dibenz[b,f][l,4]-oxazepineto fermentation in an aqueous nutrient medium containing assimilablesources of carbohydrate, nitrogen and inorganic salts, under aerobicconditions, in the presence of Cunninghamella elegans ATCC 9245.

2. A method in accordance with claim 1 in which the product is recoveredby extraction with a solvent and partition chromatography.

3. A method in accordance with claim 1 in which the product is extractedfrom the fermentation mash with n-butanol and purified by partitionchromatography.

4. A method which comprises cultivating Cunninghamella elegans ATCC 9245in an aqueous nutrient medium containing assimilable sources ofcarbohydrate, nitrogen and inorganic salts under aerobic conditions forabout 24 hours, adding 2-chloro-l l-(4- methyl 1 -piperazinyl)dibenz[b,f] [l ,4]oxazepine, continuing the fermentation for a periodof from about 60 to hours and recovering the corresponding 7- hydroxyoxazepine therefrom.

2. A method in accordance with claim 1 in which the product is recoveredby extraction with a solvent and partition chromatography.
 3. A methodin accordance with claim 1 in which the product is extracted from thefermentation mash with n-butanol and purified by partitionchromatography.
 4. A method which comprises cultivating Cunninghamellaelegans ATCC 9245 in an aqueous nutrient medium containing assimilablesources of carbohydrate, nitrogen and inorganic salts under aerobicconditions for about 24 hours, adding2-chloro-11-(4-methyl-1-piperazinyl)dibenz(b,f)(1,4)oxazepine,continuing the fermentation for a period of from about 60 to 120 hoursand recovering the corresponding 7-hydroxy oxazepine therefrom.